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Research Article

Legume Research, volume 44 issue 8 (august 2021) : 977-985

Cultural, Morphological and Molecular Characterization of Fusarium Oxysporum f.sp. Ciceris Inciting Wilt of Chickpea

K. Venkataramanamma1,*, B.V. Bhaskara Reddy1, R. Sarada Jayalakshmi1, V. Jayalakshmi1, K.V. Hari Prasad1, G. Mohan Naidu1
1Department of Plant Pathology, Shri Venkateswara Agricultural College, Tirupati-517502, Andhra Pradesh, India.

  • Submitted13-06-2019|

  • Accepted04-09-2019|

  • First Online 09-11-2019|

  • doi 10.18805/LR-4178

Cite article:- Venkataramanamma K., Reddy Bhaskara B.V., Jayalakshmi Sarada R., Jayalakshmi V., Prasad Hari K.V., Naidu Mohan G. (2021). Cultural, Morphological and Molecular Characterization of Fusarium Oxysporum f.sp. Ciceris Inciting Wilt of Chickpea . Legume Research. 44(8): 977-985. doi: 10.18805/LR-4178.

ABSTRACT

Thirty two isolates of Fusarium oxysporum f.sp. ciceris (Foc) were collected from six districts of Andhra Pradesh (A.P) and three districts of  Telangana. Based on wilting % in pathogenicity test, the isolates were grouped into three categories. All the isolates differed in cultural characters like mycelium colour, margin, texture, pigmentation and morphological characters like macro conidia, micro conidia length, width and chlamydospores diameter. There was no correlation between cultural and morphological characters of Foc isolates except three isolates (Foc-6, Foc-12 and Foc-17). These three isolates have white mycelium, cream colour pigmentation, slow radial growth and highly pathogenic (100% wilt incidence) in nature. Sporulation ability and radial growth of all isolates were also measured.  All the isolates exhibited genetic variation in RAPD banding pattern and three major clusters (I, II and III) were found in the dendrogram. Maximum genetic similarity of 80.3% was observed between Foc-27 and Foc-28 isolates.

INTRODUCTION

Chickpea is an important pulse crop of India and second most important pulse crop, after dry beans in world. It is cultivated in over 50 countries in the world.  In India, crop is grown in an area of 8.39 m.ha with a production of 7.82 m.tons with a productivity of 932 kg ha-1 (FAO, 2018).  Madhya Pradesh is the largest producer in the country accounts for over 48% of production and A.P. contributes 7% to the total production (Anonymous, 2016-17). In India, low productivity of chickpea is due to several biotic and abiotic factors and it is reported that the crop is infested by more than 52 pathogens (Nene et al., 1984). Among the diseases, Fusarium wilt caused by Fusarium oxysporum f.sp. ciceris is highly destructive and worldwide in occurrence (Nene et al., 1989). The disease can occur at all the stages of plant growth from seedling to maturity and causes an annual yield losses of 10-90 per cent (Jalali and Chand, 1992). The pathogen is soil borne, having eight races and therefore needs to know the nature of pathogen for better management of the disease. Hence, it is important to generate basic information on status, cultural, morphological and molecular variability amongst the isolates of Foc in order to have the specific breeding programme. Present study was taken up with an aim of characterising the cultural and pathogenic variability of 32 isolates of pathogen collected from A.P and Telangana and it will be helpful in establishing some correlation among cultural, morphological characters and virulence of Foc isolates.

MATERIALS AND METHODS

Isolation and identification of pathogen
 
Fusarium wilt affected chickpea plants collected from six districts of A.P. and three districts of Telangana were  used for isolation of pathogen by using tissue segment method (Rangaswamy and Mahadevan, 1999) and pure cultures were obtained by single spore culture method. The isolates were preliminarily identified as Fusarium by a key provided by Booth (1971), which included microscopic studies.
 
Pathogenecity test
 
After isolation, the isolates were tested for pathogenicity test to prove Koch’s postulates on susceptible cultivar JG-62 by using soil infestation method (Nene and Haware, 1980) to confirm it as Fusarium oxysporum f.sp. ciceris. Three replications were maintained and in each pot ten plants were maintained. In each pot number of plants wilted and days required for wilting was recorded. Based on wilting percentage, they were divided into three pathogenic groups as per the procedure of Dubey et al., (2010). These groups were, weakly pathogenic (upto 30% wilt incidence), moderately pathogenic (>30 to 50% wilt incidence) and highly pathogenic (>50% wilt incidence).
 
Cultural and morphological characterization
 
For cultural and morphological characterization the cultures incubated at 25±2°C were used and three replications were maintained for each culture and PDA medium was used. The observations on mycelium colour, pigmentation and texture were recorded in all the full grown cultures. Some cultures showed full growth in 8 days, some may attained 10 days (majority of isolates) and some grown for 12 days to occupy full plate. Radial growth of all isolates was recorded at 6 days. Morphological characters like shape, size of micro conidia, macro conidia and chlamydospores were examined in seven days cultures under calibrated compound microscope. Sporulation of all isolates was measured on haemocytometer by using compound microscope (Kumar et al., 2012). For this six mm disc of seven days old culture was used and mean of 100 observations was recorded.
 
Genetic characterization
 
Out of 32 Foc isolates, 26 isolates were selected to represent different geographical regions of A.P. and Telangana for genetic characterization through RAPD. DNA was extracted from Foc isolates by employing the SDS-lysis method given by Raeda and Broda (1985) with slight modifications. Twenty random primers belonging to K, P, OPL and OPX series were used and the list of primers were given in following table.
 
Table: Primers used for RAPD analysis.
 
Primer                                   Nucleotide sequence (5' to 3')
 
K1                                          TGCGTGCTTG
K2                                          ACTTCGCCAC
K3                                          GGCTCATGTG
K4                                          CAAACGTGGG
K5                                          CGAGGTCGACG
K6                                          CACCGCCCCAA
K7                                          GTCCTCAGTCCC
P1                                          CGTTGGATGC
P2                                          TACGGCTGGC
P3                                          GCGGCATTGT
P8                                          CAGGCCCTTC
P17                                        TACGGCTGGC
P19                                        GCGGCATTGT
P21                                        CCAGACAAGC
OPL-1                                   GGCATGACCT
OPL-07                                 AGGCGGGAAC
OPL-04                                 GACTGCACAC
OPL-13                                 ACCGCCTGCT
OPX-10                                 CCCTAGACTG
OPX-13                                 ACGGGAGCAA
 
 
These primers were used to detect polymorphism among the isolates and the following PCR conditions were followed  for amplification of DNA.
 
Stage - I         :    Initial denaturation at 94°C for 4 min
Stage - II        :    Denaturation at 94°C for 30 sec
                            Annealing at 37°C for 1 min
                           Extension at 68°C for 1 min
                           Number of cycles : 40
Stage - III       :    Final extension at 68°C for 15 minutes
 
Each amplified band was considered as RAPD marker and recorded for all samples. RAPD pattern of each isolate was evaluated by assigning character state 1 to all the bands that could be reproducible and detected in the gel and ‘0’ for the absence of the band. In order to compare the efficiency of the primers in diversity analysis, percentage of polymorphic bands and polymorphic information content (PIC) (Powell et al., 1996) were calculated.
 

Information content = 2 × fi × (1-fi)
fi: Frequency of the amplified allele (band present)1-fi: Band absent.
 
The data matrix generated was used to calculate Jaccard’s similarity coefficient for each pair wise comparison.
 
Similarity coefficient = a/n
Where,
a: Number of matching bands for each pair of comparisons.
n: Total number of bands observed in two samples.
 
These values were subjected to UPGMA cluster analysis to group the isolates based on their overall similarities and used SPSS for subsequent dendrogram preparation.

RESULTS AND DISCUSSION

A total of 32 Foc isolates were collected from wilt affected plants and among them seven from Kurnool, six from Prakasam, four each from Anantapuramu, Kadapa, Guntur, Nellore and one each from Jagtial, Adilabad and Nizamabad districts (Table 1) and the experiment was carried out from 2015-2017 at RARS,  Nandyal and IFT, Tirupati. All isolates produced aerial white mycelium on medium and identified as Fusarium based on the cultural characters and microscopic examination of micro, macro conidia, chlamydospores and they were designated from Foc-1 to Foc-32.
 

Table 1: List of Fusarium oxysporum f. sp. ciceris isolates collected from different areas of Andhra Pradesh and Telangana and pathogenicity test.


 
Further identity of Foc isolates was confirmed by pathogenicity test on   susceptible cultivar JG-62. Number of days required for expression of wilt symptom and percentage of wilt incidence were recorded for each isolate (Table 1). The isolates were highly variable in their virulence and divided into three groups such as weakly pathogenic (1 isolate), moderately pathogenic (7 isolates) and highly pathogenic (24 isolates) based on percentage of wilt incidence during pathogenicity test. Highly pathogenic isolates exhibited wilt symptoms in 20 to 32 days after sowing. Moderately pathogenic isolates exhibited wilt symptoms in 32 to 35 days and weakly pathogenic isolates exhibited wilt symptoms in 38 days after sowing. Progression of wilt symptom was increased with the age of the seedlings. These results are in accordance with Dubey et al., (2010) who categorized 112 Foc isolates into highly pathogenic (75 isolates, wilt incidence >50%), moderately pathogenic (25 isolates, wilt incidence 30-50%) and weakly pathogenic (8 isolates, wilt incidence <30%) based on  pathogenicity test on JG-62. Barhate et al., (2006) reported that one isolate out of six was highly virulent (produced 100% wilt incidence in 25 days after sowing), two isolates were moderately virulent (28 days after sowing) and some isolates produced complete wilt in 35 days after sowing. 

All the isolates differed in mycelium colour, texture, margin and pigmentation (Table 2). The cultures of different isolates exhibited variation in mycelial colour, such as dull white, white, white mycelium with sectors, whitish violet, bright white, white with light purple centre, orange white, white with light orange centre, light pinkish violet, white with light pink centre and creamish white. The texture of the mycelium in majority of isolates was fluffy in nature, in some of the isolates less fluffy, appressed, partially appressed, fluffy and thread like in nature. The appearance of margin was regular in majority of the isolates, irregular in some isolates and slight irregular in others. They were also variable with respect to pigmentation produced on medium which is of light red, creamish, creamish yellow, creamish brown,  creamish with sectors, creamish with light yellow centre, dull white with purple centre, whitish  red, pinkish, yellowish orange, light yellow, dull white, creamish white, orange yellow  and reddish brown.
 

Table 2: Cultural characters and sporulation of different isolates of Fusarium oxysporum f.sp. ciceris.


       
These results were in agreement with Nath et al., (2017) who reported that Foc isolates were highly variable in their colony growth pattern, size of colony and texture. In the present study, pigmentation of some of the isolates was changed from time to time with age of the cultures under incubation and also influenced by temperature. By visual observation, noted some pigmentation changes in some of the isolates. For ex-Foc-15 produces pink colour pigmentation and its intentisty was increased with the age of the culture. Similarly, Foc-14 showed dark brown pigmentation with the increase in its age i.e., 10 days. Hence, this character may not be appropriate for distinction of all isolates. Reddy and Dubey (2007) expressed same opinion regarding pigmentation of Foc isolates and suggested that intensity of colour varied from time to time and also depends upon period of incubation. Similarly, Burgess et al., (1989) concluded that colony morphology and pigmentation of Fusarium oxysporum were extremely variable and could not be considered suitable characters for species identification. Rodrigues and Menezes (2005) reported that environmental conditions such as growth medium, light and temperature can cause pigment production in F. oxysporum.
       
These isolates also differed in sporulation. Maximum sporulation was recorded in Foc-14 (2.2×106/ml) and minimum was in Foc-26 (0.2×106/ml). Based on sporulation ability these isolates were grouped into three categories such as abundant (11 isolates), moderate (9 isolates) and low (12 isolates) by following the procedure of Reddy and Dubey (2007). The results revealed that there was no correlation between sporulation and pathogenicity because sporulation of isolates varied within each pathogenic group. Kumar et al., (2012) observed that maximum sporulation of 1.0 × 106 conidia/ml in one isolate and minimum of 0.4 × 106 conidia /ml was in other isolate among four isolates studied and stated that sporulation has no correlation to pathogenicity.  However, in this study three pathogenic isolates Foc-6, Foc-12 and Foc-17 (cause 100% wilt incidence) and were grouped under abundant sporulation category.
       
There was a significant variation among isolates regarding radial growth. Maximum radial growth of 79 mm was observed in Foc-15, Foc-30 and these are the fast growing isolates among them. Minimum radial growth of 39 mm was observed in Foc-12. Based on radial growth pattern 16 isolates were included in first group (<60 mm of radial growth) and 16 were included in second group (60-80 mm of radial growth). Similarly, Reddy and Dubey (2007) studied cultural, morphological and radial growth at 6 days for all 25 isolates they have collected.
       
The isolates of Foc differed in the size of macro and micro conidia at microscopic level (Table 3). Among the isolates, it was observed that mean length of macro conidia was in the range of 15.02 μm (Foc-19) to 28.72 μm (Foc-9) and mean width was in the range of 2.72 μm (Foc-3) to 5.10 ìm (Foc-28). Abundant number of micro conidia was observed in all cultures. The mean micro conidia length varies from 7.1 μm (Foc-11) to 11.78 μm (Foc-28) and width varies from 2.51 (Foc-9) μm to 4.3 μm (Foc-17). Maximum diameter of (6.15 μm) chlamydospores was observed in Foc-18 and   minimum diameter (2.87 μm) was observed in Foc-27 and chlamydospores were at terminal and also intercalary in position. Kumar et al., (2012) observed chlamydospores at terminal and intercalary positions in Foc isolates.
 

Table 3: Morphological characterization of different isolates of Fusarium oxysporum f. sp. ciceris.


       
In the present research, different types of pigmentation was observed like Arvayo-Ortiz et al., (2011) who reported that  Foc strains exhibited different colours such as  whitish, brownish-white, pinkish-white to purplish-white to purplish brownish-white and the appearance of the colony varied from cottony with an abundant mycelium, patchily cottony,  cottony velvet, cottony-feathery and patchily cottony velvet. The mean length of the macro conidia was 16.5-34.1 μm, length and width means of micro conidia ranged from 7.2-9.8 × 2.5-3.3 μm and chlamydospores had a thick wall and  mean diameter ranged from 4.8-8.1 μm. These results were also in agreement with Reddy and Dubey (2007) who stated that there was no distinct correlation among radial growth, sporulation, pigmentation and size of conidia/chlamydospore with other cultural characters of Foc isolates.
       
No correlation was observed between cultural and morphological characters of Foc isolates except for three isolates (Foc-6, 12 and 17). These isolates have white mycelium, cream colour pigmentation, slow radial growth and highly Pathogenic (100% wilt incidence) during pathogenicity test. In addition, two isolates such as Foc-6 and Foc-12 have sectors on the reverse side of the colonies on PDA plates.
       
Of the twenty primers sixteen amplified and gave distinct banding pattern in RAPD analysis. Number of bands obtained was specific to each primer and ranged from 1 to 17. All the 16 primers gave a total of 1687 bands, among which 1458 (86.42%) were polymorphic (Table 4). Each primer thus produced an average of 105 bands for 26 isolates of Foc. Out of 16 primers, 9 primers such as P8, K2, K4, K5, K6, K7, P17, OPX-10 and OPX-13 (Fig 1, 2 and 3) gave 100% polymorphic bands. Least polymorphic band percentage of 43.33 was observed with P21 primer. PIC values varied from 0.16 (P21 primer) to 0.49 (K4 primer). Higher PIC values were reported for K4, P19, P17 and P2 primers and which showed higher genetic variation than other primers. Maximum number of bands (253) were produced by the P2 primer and the least (45) by P1 primer. The primer P2 yielded maximum number of 3 unique bands in Foc-12. The unique banding patterns in RAPD profile by each isolate was mainly due to the genetic differences exhibited by them. These results were in accordance with Datta and Lal (2012) who used 14 RAPD primers for genetic characterization of 14 isolates each of Foc and F.udum collected from major pulse growing regions of India and obtained 247 bands out of which 210 were polymorphic. Silva et al., (2013) reported that the PIC value varied from 0.15 to 0.28 for F. oxysporum f.sp. passiflorae by using 14 AFLP markers and used for selection of more informative markers.
 

Table 4: RAPD analysis of Fusarium oxysporum f. sp. ciceris isolates.


 

Fig 1: RAPD profiles of 26 isolates of Fusarium oxysporum f.sp. ciceris obtained with K4 primer M:Marker (1 Kb).


 

Fig 2: RAPD profiles of 26 isolates with P2 primer M: Marker (1 Kb).


 

Fig 3: RAPD profiles of 26 isolates of Fusarium oxysporum f.sp. ciceris obtained with P19 primer.


       
UPGMA cluster analysis based on RAPD profiles grouped 26 Foc isolates into three major clusters (I, II and III) (Fig 4). The cluster I comprised of eight isolates namely Foc-27, 28, 13, 9, 8, 10, 17 and Foc-31. Out of which four isolates were from Prakasam (Foc-8, 9, 10, 13) two from Kadapa (Foc-27 and 28), one each from Anantapuramu (Foc-17) and Karimnagar (Foc-31) districts. The cluster II consists of nine Foc isolates namely Foc-11, 20, 26, 14, 21, 18, 24, 15 and Foc-30. Among them 3 isolates were from Nellore (Foc-18, 21, 24) and two from Anantapuramu (Foc-14, Foc-15) and one each from Prakasam (Foc-11), Guntur (Foc-20), Kadapa (Foc-26) and Adilabad (Foc-30) districts. Seven   isolates Foc- 3, Foc-4, Foc-1, Foc-2, Foc-5, Foc-23 and Foc-22 falls under cluster III.  Out of which five isolates (Foc-1, 2, 3, 4, 5) from Kurnool and two (Foc-22 and Foc-23) from Guntur district. Cluster III has a sub cluster which has two isolates Foc-6 and Foc-12. It was observed that these clusters represented some isolates which originated from same district. Similar results were obtained by Mandhare et al., (2011) who observed high diversity among 24 isolates of Foc collected from Maharashtra and divided them into two major clusters. Cluster-I having maximum (22 no) isolates, cluster-II has two independent isolates and these clustering was more or less correlated with the geographical origin of the isolates.
 

Fig 4: Dendrogram generated by UPGMA showing the genetic diversity of Fusarium oxysporum f.sp. ciceris isolates derived from RAPD data.


       
Similarity matrix indicated that maximum genetic similarity was between Foc-27 and Foc-28 with 80.3% and the least of 24.7% was between Foc-12 and Foc-17. High level of polymorphism was observed among different Foc isolates. Datta and Lal (2012) opined that high level of genetic variability in isolates of F. oxysporum and F.udum are derived from genetically distinct clones, exchange of contaminated seeds and cultures probably contributed to existence of variable population of these pathogens in wider geographical areas. Gordon and Martyn (1997) reported that high degree of pathogenic and genetic variation in asexual fungi such as Foc might be due to the accumulation of mutations over time and somatic recombination through parasexuality in F. oxysporum (Molnar et al., 1990). Probably, these are the reasons for pathogenic and genetic differences of Foc isolates in the present study.
       
Some of these isolates possess similar cultural characters and virulence pattern and the RAPD technique could differentiated them. The isolates  Foc-6, Foc-12  and Foc-17 produced white mycelium, creamish pigmentation, slow radial growth, highly pathogenic (100% wilt incidence) and among them Foc-6 and Foc-12 were grouped under one cluster and they also have sectors on the reverse side of the plate, where as Foc-17 belongs to different cluster.  Random PCR approaches are being increasingly used to generate molecular markers which are useful for taxonomy and for characterizing fungal populations. Reddy and Dubey (2006) opined that both pathogenic virulence analysis and RAPD markers are useful tools for analysing the structure of the pathogen population, but further studies are needed to make them complementary to each other.
       
The present finding also confirmed the existence of molecular variability among the isolates of Foc. This information will be helpful for pathologists and breeders to design effective resistance breeding program in chickpea by taking into consideration about the diversity of pathogen.

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